Commercial mushrooms grow with dikaryotic mycelia; that is, cells have two nuclei present, usually of different, compatible mating types. This is beneficial, since the presence of two nuclei of different mating types is required before fruiting for spore production will occur, and commercial mushroom strains can produce the fruiting bodies needed for sale without concern about mating efficiency during the production stage.
The commercial mushroom Agaricus bisporus (also called A. brunnescens) produces heterokaryotic spores, which means the spores contain two nuclei, so mycelia grown from spores of this species can produce fruiting bodies without any need for prior mating with another strain. In other words, this mushroom species is self-fertile. The result of this reproductive strategy is inbreeding, because there is no reproductive need for sexual crossing with another strain that might have different genetic characteristics. The organism gains no advantage from mating with another organism of differing genotype, since it is capable of producing spores using the pair of nuclei already present. This ability results in a decreased incidence of genetic recombination, which in turn results in a lower number of new genotypes than are produced by other species.
Commercial strains of this mushroom have extremely few genotypes--in the work reported by Royse, et al. (Mycologia, 74:93-102, 1982), only five distinct genotypes were found among a sample of 34 commercial mushroom strains. Castle, et al. (Appl. Environ. Microbiol., 53:816-822, 1987), also found extreme limitation in the number of genotypes present in a selection of commercial mushroom strains; they even suggested that recessive lethal alleles in one of the two nuclei present, which would be masked by the alleles of the other nucleus, may account for part of the limitation in outcrossing that occurs among commercial mushroom strains.
The low level of genetic diversity within commercial mushroom strains was originally detected by Royse, et al. (1982), using isozyme analysis. By means of this analysis these workers were able to identify homokaryotic strains, those having only one nuclear type, and they were able to produce crosses between different homokaryotic strains. Castle, et al. (1987, above, and Appl. Environ. Microbiol., 54:1643-1648, 1988), extended this type of study by means of restriction fragment length polymorphism (RFLP) analysis which gave an improved ability to identify genotypes and to detect successful crosses, based on the presence of alleles from one or both putative parents.
One approach to the development of improved strains for commercial production is disclosed by Eger et al., in U.S. Pat. No. 4,242,832, where these workers proposed the isolation of a collection of monokaryotic strains, which can be characterized as to their genetic makeup and then mated in desired pairs to produce a desired genotype in the resultant dikaryote. This approach is limited by the range of genetic variability within the existing genotypes present in the strains used for mating, which is a severe limitation for the strains used in commercial mushroom production. Elliot, et al., U.S. Pat. No. 4,608,775, teaches a procedure for increasing genetic diversity by classical mutagenesis, using homokaryotic strains subjected to mutagens such as ultra-violet light, followed by mating of the mutated strains. While he was successful in a particular instance involving resistance to fungicides, work of this type is tedious and technically difficult, with limited potential for improvement within the species.
The genus Agaricus contains a number of species apart from A. bisporus, the species used for commercial mushroom production. These other species have different morphological and growth characteristics. For example A. bisporus can be morphologically distinguished from A. bitorquis, because A. bisporus has round caps, moderate length stipes and a single annular ring around the stipe, while A. bitorquis has flattened caps, short stipes and a double annulus. Whereas A. bisporus has an optimum growth temperature of 25-27 C. and a maximum growth temperature of 29-30 C., A. bitorquis has an optimum growth temperature of 30 C. and a maximum growth temperature of 34-35 C. These gross differences between the two species are, of course, the result of differences in the enzyme composition, and ultimately the genetic makeup of the species.
A potential source of genetic diversity to use in development of improved commercial mushroom strains could be the related but distinct genes of other species in the genus Agaricus. However, Anderson, et al. (Can. J. Bot., 62:1884-1889, 1984), reported that matings of homokaryotic Agaricus mushroom strains were successful within a given species, but were unsuccessful between different species of the same genus. This prevents the introduction of diversity from related species.
A method used in other organisms to introduce genetic diversity is protoplast fusion using protoplasts of two different species. The use of this technique in fungi has been reviewed by Peberdy (Microbiol. Sci., 4:108-114, 1987). First, protoplasts are prepared by enzymatically dissolving the cell walls of the fungi in a medium of high osmotic strength. Then protoplasts from two strains are mixed in the presence of polyethylene glycol and calcium ion. The fused protoplasts are transferred to a new growth medium in which normal cell morphology is regenerated. Some of the resultant cells have genetic material from both of the parent strains. In order to select for fusion products of both parent stains, these experiments are often done using parent strains which are auxotrophic for different nutrients. Subsequent use of minimal growth medium prevents regeneration of protoplasts from the auxotrophic parents, and only fusion products with complimentary genetic capability resulting in prototrophy will grow. Peberdy reports a number of crosses in yeast between different species that were achieved by means of protoplast fusion.
Successful interspecies fusion has also been reported for fungi imperfecti. Reymond, et al. (Enzyme Microb. Technol., 8:41-44, 1986), used auxotrophic strains of Penicillium in a search for improved fungi to use in cheese-making. He reported a number of successful intraspecies fusions. He also reported sucessful interspecific fusion between P. caseicolum and P. album; however interspecies fusion between P. roqueforti and either of the other two Penicillium species above was never successful.
Protoplast fusion has also been reported in basidiomycetes. Sonnenberg, et al. (Theor. Appl. Genet., 74:654-658, 1987), describes fusion of protoplasts of the basidiomycete species Schizophyllum commume using electrofusion, in place of the more common polyethylene glycol-induced fusion. Abe, et al. (Agric. Biol. Chem., 46:1955-1957, 1982), discloses a method for intrasprecies fusion of protoplasts from Tricholoma matsutake using glycine-CaCl.sub.2 -NaCl, because polyethylene glycol is reported to rupture the protoplasts, rather than stimulate fusion. Subsequently this group reported interspecies fusion of T. matsutake (Japanese Patent Document 57-029229A) in which polyethylene glycol-induced fusion was used. Yoo, et al. (Kor. J. Mycol., 14:9-15, 1986), reported interspecific fusion in Pleurotus which resulted in fruiting-capable progeny. In contrast Toyomasu, et al. (Agric. Biol. Chem., 51:2037-2040, 1987), also studying Pleurotus, found that his interspecific fusion products were incapable of fruiting unless they were back-crossed with one of the parent strains to produce a new heterokaryotic strain. Castle, et al. (1987), reports preparation of protoplasts from Agaricus, the fungal genus which includes commercial mushroom strains, but insofar as the inventor of this application is aware no one has attempted fusion, either within one species or between species, in this mushroom genus.